Which of the following technique is not use in Pyrosequencing :
A. Emulsion PCR
B. Ligation based PCR
C. Nick translation
D. Inverse PCR

Pyrosequencing Integration With PCR

Pyrosequencing is a powerful DNA sequencing method based on the principle of detecting pyrophosphate (PPi) release during nucleotide incorporation. It provides real-time sequence information by generating light signals when nucleotides are added to a growing DNA strand. Pyrosequencing is widely used in mutation detection, single nucleotide polymorphism (SNP) analysis, and DNA methylation studies.

While various molecular biology techniques are integrated into pyrosequencing, some methods are not compatible with this technology. Let’s explore the techniques involved and why certain methods are excluded from pyrosequencing.

Correct Answer:

The correct answer is C. Nick translation.

Why Nick Translation is NOT Used in Pyrosequencing

1. Principle of Nick Translation:

• Nick translation is a DNA repair mechanism where a DNA polymerase I enzyme removes nucleotides ahead of a nick (using its 5’ → 3’ exonuclease activity) and replaces them with new nucleotides.
• This technique is primarily used for labeling DNA probes or generating double-stranded DNA fragments.

2. Why It Doesn’t Work in Pyrosequencing:

• Pyrosequencing relies on DNA polymerase to incorporate nucleotides, not to remove or replace them.
• Nick translation would interfere with the detection of pyrophosphate release, which is the basis of pyrosequencing signal detection.

Explanation of Other Options

How Pyrosequencing Works

1. DNA Fragmentation and Adapter Ligation:

• The target DNA is fragmented.
• Adaptors are ligated to the fragmented DNA.

2. Emulsion PCR:

• DNA fragments are amplified using emulsion PCR.
• Each fragment is isolated in a droplet containing reagents and primers.

3. Nucleotide Incorporation and Signal Detection:

• DNA polymerase adds nucleotides to the growing strand.
• If a correct nucleotide is added, pyrophosphate (PPi) is released.

4. Light Signal Generation:

• Pyrophosphate release activates an enzymatic cascade involving ATP sulfurylase and luciferase, producing light.
• The intensity of the light signal indicates the successful addition of nucleotides.

Why Pyrosequencing is Effective

Real-time monitoring of nucleotide incorporation
High-throughput capability
 No need for labeled nucleotides or electrophoresis
 Suitable for short to medium-length sequences (100–300 bp)

Challenges in Pyrosequencing

1. Homopolymer Sequences:

• Difficult to accurately count repeated nucleotides (e.g., AAAA).

2. Limited Read Length:

• Pyrosequencing is not suitable for long DNA sequences due to signal degradation.

3. Background Noise:

• Misincorporation or incomplete extension can generate false signals.

Advantages of Pyrosequencing

1. Fast and Accurate:

• High accuracy for SNP analysis and mutation detection.

2. Quantitative:

• Can measure allele frequency and DNA methylation levels.

3. No Need for Gel Electrophoresis:

• Direct sequence detection based on light emission.

Applications of Pyrosequencing

1. Mutation Detection:

• Detection of cancer-associated mutations.

2. Epigenetic Studies:

• Methylation analysis in cancer and developmental biology.

3. Genotyping:

• Identification of single nucleotide polymorphisms (SNPs).

4. Microbial Identification:

• Sequencing bacterial and viral genomes.

Why Nick Translation Is Not Compatible with Pyrosequencing

• Pyrosequencing relies on incorporation of nucleotides and detection of pyrophosphate release.
• Nick translation involves exonuclease activity and nucleotide replacement, which would interfere with the pyrosequencing signal.
• Therefore, nick translation is not a suitable technique for pyrosequencing.

Conclusion

Pyrosequencing is a powerful sequencing technology based on real-time detection of nucleotide incorporation. Techniques like emulsion PCR, ligation-based PCR, and inverse PCR enhance the accuracy and sensitivity of pyrosequencing. However, nick translation is not compatible with pyrosequencing due to its exonuclease activity, which would interfere with the detection mechanism. Thus, option C is the correct answer.